title: Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica.
abstract: A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Taketoshi
authors.initials: T
authors.lastname: Takara
authors.forename: Tetsuto
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Masami
authors.initials: M
authors.lastname: Isobe
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.forename: Sachiyo
authors.initials: S
authors.lastname: Ichinose
authors.forename: Akira
authors.initials: A
authors.lastname: Omori
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D049874:Corbicula / Q000737:chemistry*; D006224:Cricetinae; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006736:Horses; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013109:Sphingomyelins / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins